Shu-Guang Liu

Clinical features, early treatment responses, and outcomes of pediatric acute lymphoblastic leukemia in china with or without specific fusion transcripts: A single institutional study of 1,004 patients†

Acute lymphoblastic leukemia (ALL) with distinct fusion transcripts has unique clinical features. In this study, the incidence, clinical characteristics, early treatment response, and outcomes of 1,004 Chinese pediatric ALLs were analyzed. Patients with TEL‐AML1 and E2A‐PBX1 fusion genes or other B cell precursor ALLs (BCP‐ALL) had favorable clinical features, were sensitive to prednisone, had low minimal residual disease (MRD), and an excellent prognosis, with a 5‐year event‐free survival (EFS) of 84–92%. T‐ALL was associated with a high WBC, increased age, more central nervous system involvement, a poor prednisone response, and high MRD, with a 5‐year EFS of 68.4 ± 5.2%. Patients with BCR‐ABL and MLL rearrangements usually had adverse clinical presentations and treatment responses, and a dismal prognosis, with 5‐year EFS of 27.3 and 57.4%, respectively. We also showed that BCR‐ABL and MLL rearrangements, the prednisone response, and MRD were independent prognostic factors. Interestingly, the BCH‐2003 protocol resulted in a better outcome for E2A‐PBX1+ patients than the CCLG‐2008 protocol. Intermediate and late relapses were more common in TEL‐AML1+ patients and other BCP‐ALLs compared with other subgroups (P = 0.018). Therefore, this study suggests that a fusion gene‐specific chemotherapy regimen and/or targeted therapy should be developed to improve further the cure rate of pediatric ALL. Am. J. Hematol. 2012.

Effects of methylenetetrahydrofolate reductase gene polymorphisms on toxicities during consolidation therapy in pediatric acute lymphoblastic leukemia in a Chinese population

This study aimed to investigate whether there was a correlation between the genotype or haplotype of the methylenetetrahydrofolate reductase gene (MTHFR) and toxicities during consolidation therapy or plasma methotrexate (MTX) levels at 48 h after the first dose of MTX infusion. We retrospectively genotyped 181 children with newly diagnosed acute lymphoblastic leukemia (ALL) who were treated with the Chinese Childrens Leukemia Group protocol. In standard- and medium-risk treatment branches, the 677T carriers (CT + TT) had a higher risk of developing thrombocytopenia when compared with carriers of the CC genotype (odds ratio [OR] 5.21, 95% confidence interval [CI] 1.18–23.01, p = 0.017). The 1298AC/CC genotypes were associated with a decrease in skin toxicity, as compared with the common AA genotype (p = 0.037). An estimation of haplotype frequencies showed that there was no 677T–1298C haplotype in the population. A lower frequency of anemia (OR 0.44, 95% CI 0.21–0.90, p = 0.025) and lower MTX level (p = 0.044) were observed in patients with the 677C–1298C haplotype than in those without. High plasma MTX level was correlated with anemia (p = 0.011) and neutropenia (p = 0.044). In the high-risk group, the polymorphisms or plasma MTX levels were not correlated with any toxicity. Taken together, our data demonstrate that genotyping of MTHFR and measurement of plasma MTX levels might be useful to optimize MTX therapy.

CASP8AP2 is a promising prognostic indicator in pediatric acute lymphoblastic leukemia

The prognostic significance of caspase 8 associated protein 2 (CASP8AP2) in pediatric ALL is controversial. We determined a cut-off of CASP8AP2 expression in bone marrow samples of 39 newly diagnosed patients, and found a significantly poor bone marrow relapse-free survival (p=0.019) in low-expression group and verified it in another cohort of 106 patients (p=0.002). Furthermore, as an independent prognostic factor, CASP8AP2 expression was correlated to minimal residual disease (MRD), and incorporating it with MRD would help to identify patients at greater risk of bone marrow relapse. We also developed an algorithm comprised of clinical risk and CASP8AP2 expression, which could predict bone marrow relapse more accurately.

FPGS rs1544105 polymorphism is associated with treatment outcome in pediatric B-cell precursor acute lymphoblastic leukemia

BackgroundFolypolyglutamate synthase (FPGS) catalyzes the polyglutamation of folates and antifolates, such as methotrexate (MTX), to produce highly active metabolites. FPGS tag SNP rs1544105C > T is located in the gene promoter. The aim of the present study was to investigate the impact of rs1544105 polymorphism on the treatment outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL).MethodsThis study enrolled 164 children with BCP-ALL. We genotyped the FPGS SNP rs1544105, and analyzed the associations between its genotypes and treatment outcome. We also examined FPGS mRNA levels by real-time PCR in 64 of the 164 children, and investigated the function of this polymorphism on gene expression.ResultsWe found significantly poor relapse-free survival (RFS) (p = 0.010) and poor event-free survival (EFS) (p = 0.046) in carriers of CC genotype. Multivariable Cox regression analyses adjusted for possible confounding variables showed that, relative to the CT + TT genotypes, the CC genotype was an independent prognostic factor for poor RFS (hazard ratio [HR], 4.992.; 95% CI, 1.550-16.078; p = 0.007). No association was found between any toxicity and rs1544105 polymorphism. Quantitative PCR results showed that individuals with the T allele had lower levels of FPGS transcripts.ConclusionsOur study indicates that FPGS rs1544105C > T polymorphism might influence FPGS expression and affect treatment outcome in BCP-ALL patients.

NOTCH1 mutations are associated with favourable long-term prognosis in paediatric T-cell acute lymphoblastic leukaemia: a retrospective study of patients treated on BCH-2003 and CCLG-2008 protocol in China

Activating mutations of NOTCH1 are a common occurrence in T‐cell acute lymphoblastic leukaemia (T‐ALL), but its impact on T‐ALL treatment is still controversial. In this study, the incidence, clinical features, and prognosis of 92 Chinese children with T‐ALL treated using the Beijing Childrens Hospital‐2003 and Chinese Childhood Leukaemia Group‐2008 protocols were analysed. NOTCH1 mutations were found in 42% of T‐ALL patients and were not associated with clinical features, prednisone response, and minimal residual disease (MRD) at day 33 and 78. However, proline, glutamate, serine, threonine (PEST)/transactivation domain (TAD) mutations were associated with younger age (15/16 mutant vs. 48/76 wild‐type, P = 0·018) and more central nervous system involvement (4/16 mutant vs. 3/76 wild‐type, P = 0·016); while heterodimerization domain (HD) mutations were associated with KMT2A‐MLLT1 (MLL‐ENL; 4/30 mutant vs. 1/62 wild‐type, P = 0·037). Furthermore, prognosis was better in patients with NOTCH1 mutations than in those with wild‐type NOTCH1 (5‐year event‐free survival [EFS] 92·0 ± 4·5% vs. 64·0 ± 7·1%; P = 0·003). Long‐term outcome was better in patients carrying HD mutations than in patients with wild‐type HD (5‐year EFS 89·7 ± 5·6% vs. 69·3 ± 6·2%; P = 0·034). NOTCH1 mutations and MRD at day 78 were independent prognostic factors. These findings indicate that NOTCH1 mutation predicts a favourable outcome in Chinese paediatric patients with T‐ALL on the BCH‐2003 and CCLG‐2008 protocols, and may be considered a prognostic stratification factor.

Low expressions of ARS2 and CASP8AP2 predict relapse and poor prognosis in pediatric acute lymphoblastic leukemia patients treated on China CCLG-ALL 2008 protocol

ARS2 protein is important to early development and cell proliferation, in which ARS2-CASP8AP2 interaction is implicated. However, the predictive significance of ARS2 in childhood acute lymphoblastic leukemia (ALL) is unknown. Here we evaluate the predictive values of ARS2 expression and combined ARS2 and CASP8AP2 expression in relapse. We showed that ARS2 expression in ALL bone marrow samples at initial diagnosis was markedly lower than that in complete remission (CR). Likewise, the levels of ARS2 expression in the patients suffering from relapse were significantly lower than that of patients in continuous CR. Furthermore, low expression of ARS2 was closely correlated to poor treatment response including poor prednisone response and high minimal residual disease (MRD), and the patients with high MRD (≥10(-4)) and low ARS2 were more subject to relapse. The multivariate analyses for relapse free survival and event free survival revealed that ARS2 expression remained an independent prognostic factor after adjusting other risk factors. In addition, combined assessment of ARS2 and CASP8AP2 expression was more accurate to predict relapse, based on which an algorithm composed of ARS2 and CASP8AP2 expression, prednisone response and MRD (day 78) was proposed. Together, ARS2 and CASP8AP2 expressions can precisely predict high-risk of relapse and ALL prognosis.

Polymorphisms in methotrexate transporters and their relationship to plasma methotrexate levels, toxicity of high-dose methotrexate, and outcome of pediatric acute lymphoblastic leukemia

High-dose methotrexate (HDMTX) plays an important role in the treatment of acute lymphoblastic leukemia (ALL) although there is great inter-patient variability in the efficacy and toxicity of MTX. The relationship between polymorphisms in genes encoding MTX transporters and MTX response is controversial. In the present study, 322 Chinese children with standard- and intermediate-risk ALL were genotyped for 12 polymorphisms. SLCO1B1 rs10841753 showed a significant association with plasma MTX levels at 48 h (P = 0.017). Patients who had the ABCB1 rs1128503 C allele had longer duration of hospitalization than did those with the TT genotype (P = 0.006). No association was found between oral mucositis and any polymorphism. Long-term outcome was worse in patients with the SLCO1B1 rs4149056 CC genotype than in patients with TT or TC (5-year event-free survival [EFS] 33.3 ± 19.2% vs. 90.5 ± 1.7%, P < 0.001), and was worse in patients with the SCL19A1 rs2838958 AA genotype than in patients with AG or GG (5-year EFS 78.5 ± 4.6% vs. 92.2 ± 1.8%, P = 0.008). Multiple Cox regression analyses revealed associations of minimal residual disease (MRD) at day 33 (hazard ratio 3.458; P = 0.002), MRD at day 78 (hazard ratio 6.330; P = 0.001), SLCO1B1 rs4149056 (hazard ratio 12.242; P < 0.001), and SCL19A1 rs2838958 (hazard ratio 2.324; P = 0.019) with EFS. Our findings show that polymorphisms in genes encoding MTX transporters substantially influence the kinetics and response to HDMTX therapy in childhood ALL.

E2F3a gene expression has prognostic significance in childhood acute lymphoblastic leukemia

To study E2F3a expression and its clinical significance in children with acute lymphoblastic leukemia (ALL).

MRD analysis and treatment outcome in three children with SET-NUP214-positive hematological malignancies

Sir, Chromosome aberrations that generate fusion genes are common in hematological malignancies. The SETNUP214 fusion gene resulting from balanced t(9;9) (q34;q34) or del(9)(q34.11q34.13) is rare in hematological malignancies. The SET-NUP214-positive cases were reported as patients with acute undifferentiated leukemia (AUL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myelomonocytic leukemia (von Lindern et al., 1992; Van Vlierberghe et al., 2008) with estimated incidence of 2.9% in adult T-ALL (Gorello et al., 2008). However, there is no conclusive evidence in the aspect of minimal residual disease (MRD) and prognosis for the SETNUP214-positive pediatric hematological malignancies till now. Here, we reported the dynamics of MRD and outcomes of three children with SET-NUP214-positive leukemia/lymphoma. Of the 803 children with hematological malignancies (including 534 cases of B cell precursor ALL, 48 cases of T-ALL, 149 cases of acute myeloid leukemia, 27 cases of chronic myeloid leukemia, and 45 cases of lymphoma) enrolled in Beijing Children’s Hospital (BCH) from January 2007 to October 2010, we found three (0.4%) children with SET-NUP214-positive leukemia/lymphoma by nested multiplex PCR (Pallisgaard et al., 1998). Identical fusion transcripts with a fusion between exon 7 of SET and exon 18 of NUP214 were confirmed by sequence analysis in the three patients. Morphology analysis demonstrated lymphoid characteristics in all of the three patients. However, immunophenotyping showed some differences: CD34+/CD13+/ CD33+/CD117+/CD41+, HLA-DR+/CD33+/CD117+/CD2+, CD34+/CD10+ in the three patient respectively, in addition to CD5+/CD7+/cytoplasmic CD3+ in all of the three patients. Chromosomal karyotyping only succeeded in patient 1, which showed complex chromosomal abnormality: 49,XX,+6,+8,-17,+19,+21[3]/47,XX,+14[1]/47,XX, +20[1]/45,XX,-15[1]/46,XX[11]. Finally, these three patients were diagnosed as hybrid acute leukemia (HAL), T-ALL, and T lymphoblastic lymphoma, respectively, and treated in accordance with BCH-2003 protocol for ALL medium risk (MR) according to BFM risk criteria (Table 1). We quantified MRD at six time points (TP) (Figure 1) with two types of markers. Clonal Ig/TCR rearrangements at diagnosis were analyzed using BIOMED-2 standard multiplex PCR protocols (van Dongen et al., 2003). Allele-specific oligonucleotides were designed and used in combination with the germline TaqMan probes and reverse primers to quantify MRD according to European MRD laboratory guidelines (van der Velden et al., 2007; Liu et al., 2008; Cui et al., 2010). Quantitative range of 10 and sensitivity of 10 10 were achieved in all the RQ-PCR reactions (Figure 1a). We chose TRD/TRB, TRD, and TRB rearrangement as MRD targets for the three patients, respectively. To quantify MRD with SET-NUP214 fusion transcript, we had tried to design TaqMan probe and primers but failed to reach good sensitivity and reproducibility. At last, the SYBR GREEN I chemistry was chosen for the detection of the fusion transcript. A standard curve (Slope: )3.35, R:0.99 and Intercept: 36.77) was established using serial dilutions of recombinant plasmids containing SET-NUP214 fusion fragment. The quantitative range and sensitivity were 10 copies and one copy, respectively (Figure 1b). ABL gene was used as an internal control (Beillard et al., 2003). In all of the 15 follow-up bone marrow (BM) samples, there was a strong correlation between MRD detected by fusion transcript and Ig/TCR rearrangements (Spearman r = 0.9034, P < 0.0001). This indicated that the two methods were largely consistent for each patient. So, the SET-NUP214 fusion transcript could be used as an alternative MRD marker for this type of hematological malignancy. We noted tremendous differences in MRD and outcome among the three patients. Patient 1 with HAL reached complete remission (CR) at the 5th month and underwent allogeneic HSCT after 45 days; accordingly, MRD was always above 10. She finally suffered BM relapse at the 12th month from the beginning of chemotherapy. Patient 2 with T-ALL achieved clinical CR at LETTER TO THE EDITOR INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

STAT1 single nucleotide polymorphisms and susceptibility to immune thrombocytopenia

Abstract Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder. One of the key mediators of IFN-γ signaling is the signal transducer and activator of transcription 1 protein (STAT1). We evaluated the relationship between STAT1 gene single nucleotide polymorphisms (SNPs) and the associated risk of ITP in a prospective case–control study. A total of 548 children were recruited: 328 children with ITP and 220 healthy children as sex- and age-matched normal controls. The Sequenom MassArray system (Sequenom, San Diego, CA) was used to detect three SNPs genotypes in the STAT1 gene: rs10208033, rs12693591, and rs1467199. There is a statistically significant difference in STAT1 rs1467199 allele frequencies with comparison of each of the four clinical subgroups of ITP patients to the normal controls (p = 0.0432). Also, newly diagnosed ITP patients and chronic ITP patients demonstrate significant different genotypes (χ2 = 8.511, p = 0.0142) and allelic frequency (p = 0.0055). Although a positive STAT1 rs1467199 genotype subgroups to the STAT1 mRNA expression level cannot be established, there is a weak correlation between STAT1 mRNA level and the activity ratio of Type 1 T helper lymphocyte and Type 2 T helper lymphocyte (Th1/Th2 ratio) (p = 0.0544); correlation with IFN-γ alone did not reach statistical significance (p = 0.1715). The findings in our study suggest that STAT1 rs1467199 SNP plays a potential role in the IFN-γ dependent development of autoimmunity in children with ITP. The important clinical implication of STAT1 SNPs testing as a predictor of pediatric chronic ITP will be validated in future molecular and protein functional analysis.